Coated polymer article and its use

ABSTRACT

A polymer article having structure (I), where P comprises the basic polymer and groups —L—CH(—X(R 1 ) p )—CH 2 (—Y(R 2 ) p ); L is a part of a pending group utilized for introducing —X(R 1 ) p  and —Y(R 2 ) p ; X and Y are halogen, N, S and O; R 1  is hydrogen, alkyl, acyl or R 2  when X is N, O or S; R 2  is hydrogen, alkyl, alkylaryl or arylalkyl in which the alkyl part may contain  1-18  carbons, or —R 3 (—NH—CR 4 ═O) q  or —R 3 (—NH 2 ) q , —CR 4 ═O or poly alkyloxy that may have been terminally acylated or alkylated; p is an integer  0-3,  with the provisos a) p depends on X being halogen, O, S or N, and b) that if several groups R 2  are present they may be identical or different; R 3  is alkyl, —O-alkyl, hydroxyalkyl, phenylalkyl, with up to  18  carbon atoms in the alkyl part, and R 4  is C 1-18  alkyl. q is an integer&gt;O representing that one or more hydrogens in R 3  may have been replaced with —NH 2  or —NH—CR 4 ═O. The polymer article may be used as a chromatography separation medium or in solide phase oligonucleotide synthesis.

TECHNICAL FIELD AND BACKGROUND ART

The present invention relates to coated polymer articles and processesfor the production thereof and use of the article as chromatographymedium. Specifically the invention relates to coated articles with thecoating covalently bound to the polymer surface via pending double bondsof the polymer surface. An example of pending groups are residual vinylgroups remaining after polymerisation of vinyl monomers, such as divinylbenzene monomers.

In the context of the present invention the term “coated”/“coating”means that pending alken groups on the surface has been derivatized tointroduce novel functionaities that may be adapted to two specific usesof the article (chromatographic support and support for oligopeptide andoligonucleotide synthesis). “Coat” thus has a somewhat different meaningcompared to coating in the sense of physical adsorption andstabilization by crosslinking of an adsorbed layer (coat) within thelayer and/or to the original surface.

The stationary phase (support, matrix) in liquid chromatography iscomposed of a porous matrix and is most commonly in bead form. A widevariety of materials are used for chromatography matrices, bothinorganic and organic materials. The demands on the matrix are several.It should be chemically and physically stable and be able to withstandextreme pH conditions. The matrix should be rigid to allow high flowrates in columns packed with particles of small diameters. It must alsobe possible to produce particles with a broad range of porosities.

The surface characteristics of the matrix is important. By introductionof new chemical structures on the surface of the matrix it is possibleto design stationary phases which interact more or less specificallywith a particular molecule. In chromatographic separation of proteins inaqueous solution the matrix is usually hydrophilic. In reversed phasechromatography (RPC) for peptide or protein separation, silica gels havebeen used as chromatography medium. Silica gels are rigid enough towithstand high flow rates and different hydrophobicities are available(C4, C8, C18). However, silica gels are not stable above pH 8.0 andhence can not be cleaned by using alkali. In stead it is common to userigid matrices of organic polymers e.g. polymethacrylate orpolystyrene/polystyrene-divinylbenzene. Polystyrene-divinylbenzeneparticles are hydrophobic in aqueous solutions and large amounts oforganic solvents have to be used to eluate the absorbed molecules. Theselectivity is also limited. Due to the fact that there is only a smallvariation in hydrophobicity of these particles, the use of them islimited. In contrast the ratio hydrophobicity/hydrofilicity of silicaparticles can be varied by chemical reactions. There are a large varietyin chemistry available for modification of the hydrofilic silicasurface. To increase the possibilities to use particles ofpolystyrene-divinylbenzene and other hydrophobic polymers, it isrecognized that the surface of the particles must be modified to be morehydrophilic before they can be used as chromatographic separationmedium. Different methods have been suggested for hydrophilisation ofthe surface of particles based on hydrophobic polymers:

WO 91/11241 relates to a method of producing a hydrophilic coating on ahydrophobic surface by which a compound comprising a hydrophobic and ahydrophilic domain is adsorbed on the surface. The draw backs with thismethod are several. The polymer to be adsorbed has a high molecularweight and therefore the polymer will not spread out evenly in allpores. The control of the pore size distribution is then lost and somepores may not even be coated at all. As the polymer coating is onlyadsorbed it may loosen from the surface at contacting with non-polarsolvents. Therefore the coating must be cross-linked.

WO 95/23022 discloses a method of covalently bonding a hydrophiliccoating on a hydrophobic surface. An unsaturated polymer coating isgrafted to the surface via the unsaturated groups on the surface. Alsowith this method there is the same problem of obtaining the coatingevenly distributed on the porous surface of the matrix.

Various uses of vinyl groups pending on vinyl benzene polymers have beenreviewed during the priority year by Hubbard et al (Reactive FunctionalPolymers 36 (1998) 1-16 and 17-30). The use of pending vinyl groups forimmobilizing a catalyst on a macroporous polystyrene resin has beendescribed by Faber et al (Reactive Polymers 11 (1989) 117-126. Modifyingchromatographic vinyl-benzene based supports by routes other than viaresidual vinyls has been described by Sun et al (J, Chromatog, 522(1990) 95-105). Still another route has been described by Moberg et al(Reactive Polymers 15 (1991) 25-35).

OBJECTIVES OF THE INVENTION

The object of the present invention is to obtain improved coated polymerarticles and improved methods of coating a polymer article to change thesurface characteristics of the article without the draw backs of knownmethods.

A further object of the invention is to produce polymer particles withsuitable surface characteristics for chromatographic use.

It is a further object of the invention to provide a coating which maybe derivatized to produce a wide range of different functional groupsfor use in different types of chromatography. The intention is to beable to achieve a large variety in both hydrophobicity/hydrophilicityand selectivity and to reduce the amount of solvent used in the elution.

The demands on supports to be used in the synthesis of oligonucleotidesand oligopeptides with respect to the balance between hydrophobicity andhydrophilicity are less critical than for chromatographic matrices,although it remains that maintaining open pores is important in order tomaintain high capacity of a given porous support. The primary object inthis aspect of the invention is to provide a simple well-defined way ofintroducing a handle in form of a primary or secondary amino group intoa polymer support based on a hydrophobic polymer.

THE INVENTION

The objects of the invention are achieved by the polymer articles andmethods defined in the claims. According to the invention a coatedpolymer article is obtained, which article is characterized in that thecoating is covalently bound to the polymer surface via double bonds ofthe polymer. The article has the structure:

 PLCH—CH₂Y(R₂)_(p)

X(R₁)_(p)

where

P comprises the basic polymer structure of the article before coatingand further groups

—L—CH(—X(R₁)_(p))—CH₂(—Y(R₂)_(p));

L is a part of a pending group projecting from the basic polymer andcomprising the double bonds utilized for introducing —X(R₁)_(p) and—Y(R₂)_(p). It may be a single covalent link to the basic polymer or abenzene ring.

X and Y are independently selected from halogens, in particular Br, andN, S and O.R₁ is hydrogen, a straight, branched or cyclic alkyl or acylgroup, such as C₁-C₁₀ alkyl, C₁-C₁₈ acyl, or R₂ when X is N, O or S withparticular emphasis for X equals Y equals O.

R₂ is hydrogen, straight, branched or cyclic alkyl, such as C₁-C₁₈alkyl, —R₃(—NH₂)_(q), alkylaryl or arylalkyl in which the alkyl part maycontain 1-18 carbon atoms,

—R₃(—NH—CR₄═O)_(q), —CR₄═O, or poly lower alkyloxy groups that may havebeen terminally acylated or alkylated (e.g. —((CH₂)_(n)—O)_(n).OR′ wheren is an integer 2, 3 or 4 and n′ is an integer 2-100 and an H in CH₂ mayhave been replaced with methyl and R′ may be any one of the expresslymentioned R₁ groups.

p=0 when Y=Br. p may be 1 when Y=O or S. p may be 2 when Y=N or S. p maybe 3 for Y=N. In case there are more than one R₂, they may be identicalor different.

R₃ is a straight, cyclic or branched alkyl, —O—alkyl, hydroxyalkyl,phenylalkyl, with up to 18 carbon atoms in the alkyl part.

R₄ is a straight, branched or cyclic alkyl group, such as C₁-C₁₈ alkyl.

q represents that one or more (1, 2, 3, 4 etc, i.e. 1-poly) hydrogens ina basic alkyl group R₃ may have been replaced with a respective primaryamino group (—NH₂) or a respective —NH—CR₄═O group.

In the above-mentioned alkyl and acyl groups, one or more hydrogens maybe optionally substituted with an amino or a hydroxy or an alkoxy groupand carbon chains may be optionally broken at one or more locations byan ether oxygen or an amino nitrogen, the proviso being that there areat most one atom selected among oxygen and nitrogen binding to one andthe same sp³-hybridised carbon atom. Among particularly interestingalkyl groups with broken hydrocarbon chains may be mentioned thosecomprising repetitive alkylene oxide units, such as —CH₂CH₂—O—,—CH₂CH₂CH₂—O—, —CH₂CH(CH₃)—O— etc.

According to a further aspect of the invention a method of producing acoated polymer article is provided, characterized in derivatizing doublebonds linked to the basic polymer structure (P) of the article throughthe group L.

According to yet another aspect of the invention a coated polymerarticle according to the invention is used as chromatography medium oras a support for the solid phase synthesis of an oligopeptide or anoligonucleotide.

With the present invention it is possible to apply a coating to anypolymer article having double bonds on the surface and thus to modifythe surface in a predetermined manner. Preferably the method is used tomanufacture chromatographic media. It was found that a wide range ofchromatography media can by synthesised by introducing differentchemical structures on a porous polymer surface. A controlled amount ofa desired group and/or charge can be introduced on the surface. With themethod according to the invention the coating, including the desiredgroup and/or charge, is evenly spread out over the surface also in thepores and then the pore size distribution is kept intact. Besides, thepore size distribution can be varied by varying the length of the groupsintroduced on the surface. The method is suitable for the production ofreversed phase chromatography (RPC) media with the same performance assilica media but without the draw backs of silica. New RPC media areprepared by introducing polar groups containing long alkyl or amidegroups close to the surface. By varying the length of the alkyl andamide chains, different matrices with various hydrophobicities and poresize distributions can be synthesised. Also primary and secondary aminogroups may be introduced which are essential for the use of support inthe synthesis of oligonucleotides.

According to the invention the double bond of a pending group istransformed to an halohydrin or vicinal halide, i.e. —CHX₁—CHY₁—, whereat least one of X₁ and Y₁ is a substituent selected from halogens, inparticular bromine, and the remaining group of X₁ and Y₁ is hydroxy oralkoxy, or to an epoxide or to corresponding nitrogen or sulphuranalogues.

Halogen may be introduced by addition of certain halogen containingcompounds, with the preferred halogen being bromine, to the double bondspresent on the polymer surface. Bromine can be introduced by knownmethods such as reaction with Br₂ in a suitable solvents, or with N—Br—succinimide in a suitable solvent. The synthesis will now be illustratedby discussing the reaction with bromine. This does not exclude the useof other halogens, such as chlorine and iodine, which often undergoanalogous reactions.

Preferably, in the first step of the method a polymer compound one ormore double bonds of a polymer (P—L—CH═CH₂ with P and L having the samemeaning as above) is reacted with Br₂ in a solution containing R₁OH or amixture of H₂O and an organic solvent soluble in H₂O, whereby a coatedpolymer article having the structure

or a mixture of the structures: (1) and

are obtained, where

R₁ is hydrogen or a straight, branched or cyclic alkyl group containingoxygen atoms as defined above. Particularly R₁ may be hydrogen or C₁-C₁₀alkyl.

As mentioned above the method according to the invention can be used onpolymer surfaces with free double bonds on the surface. As such polymercan be mentioned polymers from aromatic compounds containing vinylicgroups. The aromatic compounds can be mono-functional, di- or poly-functional. The polymer should have a sufficient amount of di- or polyvinylic groups to result in a cross-linked structure. As suitablearomatic compounds can e.g. be mentioned styrene, divinylbenzene,vinylbenzene, vinylbenzylchloride, etylvinylbenzene, acetoxyvinylbenzeneand other derivatives of vinylbenzene. In a preferred embodiment thebasic polymer structure is a cross-linked polymer of styrene,divinylbenzene and ethylvinylbenzene or a polymer of divinylbenzene andethylvinylbenzene. The pending groups are then benzene groups resultingfrom vinyl groups which have not been crosslinked. The structure can bedescribed as follows:

The free double bonds from the vinyl groups on the surface of thepolymer are used for the reaction with bromine.

In preferred embodiments of the invention the coated articles areparticles. Preferably the coated particles are prepared by coatinguncoated particles-with a diameter of 1-200 μm, preferably 3-50 μm. Thesurface area of the uncoated particles lies within the range 1-1000m²/g, preferably within 100-800 m²/g, and suitable pore volumes are from0.1-3.0 ml/g. The content of residual vinyl groups in the beads arenormally 0.3-7.7 mmol/g and should preferably be 0.3-3 mmole/g, mostpreferably 1-2 mmole/g.

In the first step of the invention the uncoated polymer article iscontacted with bromine dissolved in the same solvent. Often the polymerarticle is in the form of particles which for coating purposes issuspended in the same solvent as bromine. In the reaction with brominethe choice of solvent is important for the outcome of the reaction. Thereaction with bromine can result in a monobromide or in a dibromide orin a mixture thereof, as described above. Usually a mixture is obtainedwith a varying content of mono- and di-bromide. This content depends onthe solvent used in the process. It is known that bromination innon-polar solvents like tetrachloromethane results in a high content ofdibromide. In polar solvents like the lower alcohols, i.e. straight orbranched C₁-C₁₀ alcohols, a high content of the monobromide is obtained.However, in water the amount of dibromide achieved is high due to thefact that bromine is partly soluble in water. If water is mixed with anorganic solvent, soluble in water, and in which bromine dissolves, thenthe monobromide is obtained to a large extent. With longer alkyl groupsthe alcohols become more non-polar and then the amount of dibromideincreases. Generally it can be said that if the polarity, expressed bythe dielectric constant ∈, for the solvents or solvent mixtures is low,a high content of dibromide will be obtained. If ∈ is high theproduction of the monobromide will be favoured. In the present inventiona high amount of the monobromide is preferred. Then, ∈ between 6 and upto about 78 is preferred. Solvents suitable for the invention are C₁-C₁₀alcohols such as methanol, ethanol, 1-propanol, 1-butanol, 1-hexanol. Ifa mixture of water and an organic solvent is used, solvents liketetrahydrofuran, lower alcohols are suggested. In this latter case R inthe formula above will be H. There are many reports on the brominationreaction of vinyl groups. The following references can be mentioned:Yates, K, McDonald, R. S, and Shapiro., S. A., (1973 ): J. Org. Chem.38, 2465; J. H. Rolston and K. Yates, (1969 ): J. Am. Chem. Soc.91,1469; R. C. Fahey, (1989 ): Topics in stereochemistry, Wiley, N.Y., p280-286.

The reaction with bromine is suitably made at a temperature between−20-50° C., preferably 0-20° C. The amount of bromine used is suitably0.1-10 moles bromine/mole vinyl, preferably 0.5-2 moles bromine/molevinyl. The amount of monobromide obtained is also dependent of thebromine concentration. Concentrations of 0.05-0.5 moles/l solvent can beused, preferably 0.1-0.2 moles/l.

In the next step of the invention the brominated articles of theinvention are reacted with a compound Y(R₂)_(p) whereby a coated polymerarticle having the structure

or a mixture of the structures (3) and

are obtained, where

Y=N, S, O, and R₂, R₃ and p have the same meaning as above.

The compound Y(R₂)_(p) can be chosen from the group: ammonia, mono-di-or poly-alkylamines, mono-di- or poly-alkylalcohols, mono-di- orpoly-alkylthiols, with the amine-, alcohol-, or thiol group respectivelybeing a primary group. The alkyl group can be linear or branched and thelength of the alkyl group is preferably not more than C₁₈ in order tocomply with solubility requirements for the preferred solvents. Thelonger the alkyl group is the more hydrophobic the chromatographic mediawill be. Longer alkyl groups will also result in a reduction of smallsize pores (<50 Å in diameter) of the polymer article.

In the second step an aqueous suspension of the brominated articles ismixed with e.g. the amine. It is also possible to use a mixture of waterand a lower alcohol like methanol or ethanol as suspension medium. Thereaction temperature is suitably 50-100° C. and the reaction time about24 h.

If the brominated articles are reacted with ammonia or a primary amine(mono, di or poly), coated polymer articles having the structure:

or a mixture of structures: (5) or a mixture of structures: (5) or

or the structure:

or a mixture of structures: (16) and

are obtained, where R₁, R₂, R₃ and q have the same meaning as above.These compounds can be further reacted with an acid chloride, acidanhydride or isocyanate:

or whereby a coated polymer article having the structure

or a mixture of the structures: (9) and

or having the structure:

or a mixture of the structures: (11) and

or a coated polymer article having the structure:

or a mixture of the structures: (18) and

or the structure:

or a mixture of structures: (20) and

are obtained where R₁, R₂, R₃, and R₄ and q have the same meaning asabove and Z is halogen, such as chloride.

The reaction of the amine substituents with an acid chloride, acidanhydride or isocyanate is made in a water free organic solvent such asacetone, or another suitable industrial ketone. The solvent must notcontain groups capable of reacting with the acid chloride/anhydride orisocyanate. It is possible to use water if the temperature is kept below15° C. During this reaction HCl is produced if an acid chloride is used.HCl can be neutralised with a tertiary amine, likedi-isopropyl-ethylamine. The reaction is carried out at a temperature of20-50° C. and the reaction time suitably 1-3 h.

As an alternative to this last reaction step and if the substituent R₁is H, both the amine substituent and the hydroxyl group of formulae 5,6, 16 and 17 may be reacted with an acid chloride, an anhydride or anisocyanate whereby a coated polymer article having the structure:

or a mixture of the structure: (14) and (10) or having the structure

or a mixture of structures: (15) and (12) or having the structure:

or a mixture of structures: (23) and (19), or having the structure:

or having a mixture of the structures: (24) and (21) are obtained.

The double bonds of the pending groups may also be converted to epoxidegroups or corresponding sulphur or amino analogous by techniques wellknown in the art. For halohydrins or vicinal dihalides a properadjustment of pH may result in epoxides, by doing this in the presenceof the appropriate sulphur and amino compounds, the respective sulphurand nitrogen analogue will form. These three-membered rings will then beprone to undergo nucleophilic attack by the same compounds as outlinedabove for halohydrins and vicinal dihalides. The resulting products maythen be further derivatized, also as outlined above. An alternative forobtaining epoxides is to react the double bond with a peroxy compound.

Further the introduced groups (coat) may then be further derivatized forinstance to introduce charged groups such as —SO₃ ⁻ (sulphonate), —PO₃²⁻ (phospohonate), —O—PO₃ ²⁻ (phosphate), charged ammonium groups(primary, secondary, tertiary and quaternary ammonium groups) (freevalences bind to saturated or unsaturated carbon).

The coated polymer articles can be used as chromatography media ofdifferent types. By varying the grade of halogenation, for instancebromination, the type and amount of e.g. amine and the type and amountof acid halide, such as acid chloride, or isocyanate it is possible tomanufacture particles with a great variety in surface characteristics.Thus, with the invention it is possible to tailor the surfacecharacteristics for the different molecules that are to bechromatographically separated. According to a preferred embodiment ofthe invention the coated articles are used as RPC media.

The introduction of primary or secondary amino groups will enable thatthe inventive article can be used in the solid phase synthesis ofoligonucleotides and oligopeptides.

The invention will now be illustrated by the following examples whichhowever are not intended to limit the invention. With parts and percentare meant parts by weight and percent by weight if not explicitly statedotherwise.

In the reactions divinylbenzene particles are used. The particles havethe following characteristics: Particle size: 15 μm; surface area: 714m²/g; pore size: 2.2.ml/g; 1.5 mmol vinyl groups/g. Starting from theseparticles, eleven different coated particles for RPC were synthesised byvarying the length of the alkyl and amide chains. The obtained coatedparticles were tested by separation of peptides and compared with theuncoated particle and with a conventional silica particle, Kromasil™ C18(from Eka Nobel AB).

Experimental Part EXAMPLE 1

100 g of divinylbenzene particles in ethanol (amount of dry substance10.6%) were washed with ethanol and added to a reactor and cooled to 15°C. 25.50 g of bromine (1.60 mmol/g particles) were dissolved in 400 gethanol and added in portions over a time period of 15 minutes. Theparticles were washed with ethanol after two hours.

EXAMPLE 2

31.18 g hexylamine were added to 199.90 g aqueous suspension (drycontent: 20.0 g) of the brominated particles from example 1. Heating to90° C. was started at the same time. After 24 hours 300 ml ethanol m/10%isopropanol were added during a period of 10 minutes. The suspension wasagitated for 25 minutes and then filtrated and washed with 0.8 l water,1.6 l 0.1 M NaOH and 2.0 l water.

EXAMPLE 3

43.51 g hexadecylamine were added to 120.0 g aqueous suspension (drycontent: 12.0 g) of the brominated particles from example 1. Heating to90° C. was started at the same time. After 24 hours 180 ml ethanol m/10%isopropanol were added during a period of 10 minutes. The suspension wasagitated for 25 minutes and then filtrated and washed with 0.4 l ethanolm/10% isopropanol, 0.48 l water, 1.0 l 0.1 M NaOH and 1.2 l water.

EXAMPLE 4

7.5 ml lauroyl chloride and 0.95 ml diisopropylethylamine were added to90.13 suspension (dry content: 9.0 g) of the particles from example 2 indry acetone at ambient temperature. After 2 hours the suspension wasfiltered and washed with 360 ml acetone and 360 ml ethanol.

EXAMPLE 5

4.35 ml capryloyl chloride and 0.95 ml diisopropylethylamine were addedto 90.0 g suspension (dry content: 9.0 g) of the particles from example2 in dry acetone at ambient temperature. After 2 hours the suspensionwas filtered and washed with 360 ml acetone and 360 ml ethanol.

EXAMPLE 6

4.25 ml lauroyl chloride and 0.55 ml diisopropylethylamine were added to51.0 g suspension (dry content: 5.10 g) of the particles from example 3in dry acetone at ambient temperature. After 2 hours the suspension wasfiltered and washed with 200 ml acetone and 200 ml ethanol.

EXAMPLE 7

2.45 ml capryloyl chloride and 0.55 ml diisopropylethylamine were addedto 50.96 g suspension (dry content: 5.10 g) of the particles fromexample 3 in dry acetone at ambient temperature. After 2 hours thesuspension was filtered and washed with 200 ml acetone and 200 mlethanol.

EXAMPLE 8

16.65 g of 1,3 diaminopropane (15 mmol/g particles) were added to 15 gof the particles from example 1, in water (149.5 g suspension). Themixture was heated to 90° C. for 25 hours and 40 minutes and then washedwith water, NaOH (0.5 M), water, 20% ethanol and acetone.

EXAMPLE 9

6.00 g of the aminated particles from example 8 were washed with dryacetone. Capryloyl chloride (9.18 g, 9.4 mmol/g particles) andN-ethyldiisopropylamine (1.10 g, 1.4 mmol/g particles) were added to 60g of acetone suspension. After two hours in room temperature theparticles were washed with acetone and ethanol.

EXAMPLE 10

33.37 g of 2,2-ethylenedioxydiethylamine (15 mmol/g particles) wereadded to 15 g of the particles from example 1 in water (150.20suspension). The mixture was heated to 90° C. After 24 hours theparticles were washed with water, NaOH (0.5 M), water and acetone.

EXAMPLE 11

26.38 g of hexanediamine (15 mmol/g particles) were added to 15 g of theparticles from example 1 in water (149.97 g suspension). The mixture washeated to 90° C. After 24 hours the particles were washed with water,NaOH (0.5 M), water and acetone.

EXAMPLE 12

6.00 g of the aminated particles from example 8 were washed with dryacetone. 9.55 g (7.3 mmol/g particles) lauroyl chloride and 1.11 g (1.4mmol/g particles) N-ethyldiisopropylamine were added to 60 g acetonesuspension. After 2 hours at room temperature the particles were washedwith acetone and ethanol.

EXAMPLE 13

6.00 g of the aminated particles from example 10 were washed with dryacetone. 9.21 g (7.0 mmol/g particles) lauroyl chloride and 1.11 g (1.4mmol/g particles) N-ethyldiisopropylamine were added to 60 g acetonesuspension. After 2 hours at room temperature the particles were washedwith acetone and ethanol.

EXAMPLE 14

6.00 g of the aminated particles from example 10 were washed with dryacetone. 5.68 g (5.8 mmol/g particles) capryloyl chloride and 1.10 g(1.4 mmol/g particles) N-ethylisopropylamine were added to 60 g acetonesuspension. After 2 hours at room temperature the particles were washedwith acetone and ethanol.

EXAMPLE 15

6.00 g of the aminated particles from example 11 were washed with dryacetone. 9.20 g (7.0 mmol/g particles) lauroyl chloride and 1.10 g (1.4mmol/g particles) N-ethyldiisopropylamine were added to 60 g acetonesuspension. After 2 hours at room temperature the particles were washedwith acetone and ethanol.

EXAMPLE 16

6.00 g of the aminated particles from example 11 were washed with dryacetone. 5.65 g (5.8 mmol/g particles) capryloyl chloride and 1.10 g(1.4 mmol/g particles) N-ethylisopropylamine were added to 60 g acetonesuspension. After 2 hours at room temperature the particles were washedwith acetone and ethanol.

EXAMPLE 17

10 g of the porous particles in methanol (dry content 12.4%) were addedto a reactor and cooled to 15° C. 2.53 g of bromine (1.58 mmol/gparticles) were added in portions with a glass syringe over 15 minutes.The particles were washed with methanol.

EXAMPLE 18

15.93 g of hexylamine (17 mmol/g particles) were added to 9.14 g of theparticles from example 17 in water (91.0 g suspension). The mixture washeated at 90° C. for 24 hours, and then washed with water, sodiumhydroxide (0.5 M), water, methanol and acetone.

EXAMPLE 19

7.00 g of the aminated particles from example 18 were washed withacetone. 5.18 g (3.5 mmol/g particles) dodecylisocyanate were added to62 g acetone suspension of the particles and heated, first at 30° C. for1 hour 35 minutes, and then at 50° C. for 1 hour. The particles werewashed with acetone and ethanol.

The obtained final particles were used in chromatografic testing byseparation of peptides accordingly:

EXAMPLE 20

Separation of Peptides I

A peptide mixture consisted of (Ile⁷)-Angiotensin III,(Val⁴)-AngiotensinII, AngiotensinIII and AngiotensinI.

Sample:

0.125 mg/ml Ile⁷)-Angiotensin III

0.125 mg/ml (Val⁴)-AngiotensinII

0.125 mg/ml AngiotensinIII

0.125 mg/ml AngiotensinI

The peptides are dissolved in Solution A.

Solution A: 0.1% trifluoro-acetic (TFA) acid in water. 1 ml TFA isdissolved in 1000 ml deionized water.

Solution B: 60% acetonitrile/0.1% trifluoro-acetic acid (TFA) 0.600 mlacetonitrile was dissolved in 400 ml deionized water and 1 ml TFA.

Gradient:

0-2 col. vol. 15%B

2-22 col. vol. 15-65%B

22-25 col. vol. 65%B

25-27% col. vol. 15%B

Injection volume: 25 μL

Linear velocity: 300 cm/h

Detector: UV 214 nm, 0.2 AU

Peptides Sequence Supplier (Ile′) - Arg-Val-Tyr-Ile-His-Pro- SigmaAngiotensin III Ile A-0911 (Val⁴) - Arg-Val-Tyr-Val-His-Pro- SigmaAngiotensin III Phe A-6277 Angiotensin III Arg-Val-Tyr-Ile-His-Pro-Sigma Phe A-0903 Angiotensin I Asp-Arg-Val-Tyr-Ile-His- SigmaPro-Phe-His-Leu A-9650

Peptide Separation II

The angiotensins were all positively charged at this pH and applied as amixture. The retention time (t_(R)), capacity factor (k) and selectivity(separation factor α) were determined.

Column: Glass column Pharmacia HR 5/5 (vol.:1 ml)

Mobile phase A: 0.1% TFA

Mobile phase B: 0.1% TFA/60% acetonitrile

Gradient: 9-39% acetonitrile in 20 minutes

Injection: 25 μl of 0.5 mg/ml angiotensin mixture

Flow rate: 5 cm/min (1 ml(min)

Detector: 214 nm, 0.2 AU

System: FPLC® (Chromatography system from Pharmacia Biotech)

Before applying the sample a gradient was run on every column, and thecolumns were equilibrated with 10 column volumes of 15% mobile phase B(9% acetonitrile).

Separation of Peptides III

The angiotensins were separated due to their differences inhydrophobicty in an ascending gradient of acetonitrile.

The results are presented in the tables below and in the chromatograms.

The retention time t_(R) is the time between the start of elution andthe emergence of the peak maximum. The retention time is presented intable 1. The retention volume V_(R) is the corresponding volume ofmobile phase between the start of elution and emergence of the peak.

The separation factor α is a measure of how well two peaks areseparated. It is given by the ratio of the capacity factors of the twopeaks(k₂/k₁). The separation factor is presented in table 2.

TABLE 1 Acid chloride/ Media Ile7 tR Val4 tR AIIII tR AI tR AminesIsocyanate Example 4 7.6 8.11 9.23 11.12 Hexylamine Lauroyl- chlorideExample 5 7.1 7.79 8.87 10.62 Hexylamine Capryloyl- chloride Example 67.83 8.35 9.51 11.45 Hexadecyl- Lauroyl- amine chloride Example 7 7.538.07 9.2 11.08 Hexadecyl- Capryloyl- amine chloride Example 9 5.1 5.997.14 9.12 1,3-diamino- Capryloyl- propane chloride Example 12 5.82 6.487.7 9.65 1,3-diamino- Lauroyl- propane chloride Example 13 4.94 5.827.16 9.42 2,2-ethylene- Layroyl- dioxy-diethyl- chloride diamine Example14 3.88 5.1 6.58 8.78 2,2-ethylene- Capryloyl- dioxy-diethyl- chloridediamine Example 15 6.02 6.68 7.98 10.15 1,6-diamino- Lauroyl- hexanechloride Example 16 5.52 6.35 7.61 9.62 1,6-diamino- Capryloyl- hexanechloride Example 19 6.99 7.59 8.74 10.58 Hexylamine Dodecyl- isocyanatUnmodified 9.16 9.91 11 12.69 none none Kromasil 10.68 10.95 12.07 13.49none none C18

TABLE 2 αIle7- αVal4- αAIII- Media Val4 AIII AI Ex. 4 1.07 1.14 1.20 Ex.5 1.10 1.14 1.20 Ex. 6 1.07 1.14 1.20 Ex. 7 1.07 1.14 1.20 Ex. 9 1.171.19 1.28 Ex. 12 1.11 1.19 1.25 Ex. 13 1.18 1.23 1.32 Ex. 14 1.31 1.291.33 Ex. 15 1.11 1.19 1.27 Ex. 16 1.15 1.20 1.26 Ex. 19 1.09 1.15 1.21Unmodi- 1.08 1.11 1.15 fied Kroma- 1.03 1.10 1.12 sil C18

The modified media are all more hydrophilic than the unmodified mediaand also more hydrophilic than the silica media used for comparison. Theretention times are related to the polarity of the synthesised media.The lower the retention times, the lower the concentration of the samplein the stationary phase, the more polar the surface. The mediasynthesised using lauroyl chloride have higher retention times thanthose where capryloyl chloride were used. The most polar media weremedia prepared with diamines and capryloyl chloride according to example9 and example 14. This is evident from table 1 where the tR values arethe lowest for example 9 and 14. In accordance with this the peaks inthe chromatograms appears first in example 9 and 14. As can be seen fromthe chromatograms all peaks from the examples according to the inventionappears before the peaks for the unmodified medium and the silica mediumindicating that the particles according to the invention are morehydrophilic. Thus, the surface modifications according to the inventionhas a pronounced effect on the concentration of acetonitrile used forelution. A lower amount of acetonitrile used for elution leads to largesavings in the cost of organic solvents.

The difference in selectivity factors shows that the separation betweenthe peptides can easily be changed by the surface modifications so thata separation problem can be optimised by small changes in the productionmethod.

The media synthesised from monosubstituted amines have lower separationfactors than those synthesised from disubstituted amines. The diaminomedia were separating (Ile⁷) Angiotensin III and (Val⁴) Angiotensin IIImuch better and gave larger variation in the separation than the mediabased on monoamines.

The media according to example 9 and 14 had also the highest separationfactor for (Ile⁷) Angiotensin III and (Val⁴)Angiotensin III which isevident from table 2 and the peak separation in the chromatograms.Generally it can be seen from the chromatograms that the separation withthe media according to the invention is also much better than with theunmodified medium or the silica medium. Kromasil does not separate thepeptides properly as the peaks are not separated.

During the priority year we have shown that coatings in which the alkylgroups of R₁ and/or R₂ are repetitive units of lower alkenyloxy groups,such as —CH₂CH₂—O—, —CH₂CH₂CH₂—O—, —CH₂CH₂(CH₃)—O—, and have molecularweights up to at least 5000 dalton will function in chromatographicapplications.

During the priority year we have also introduced amino groups for makingthe beads suitable for solid phase oligonucleotide synthesis.

Addition of bromine: 10 gram of ice were washed with 300 ml 0.5 M sodiumacetate buffer pH 5.62 g of poly(divinylbenzene-ethylvinylbenzene)particles in sodium acetate buffer pH 5.0 were transferred to a 100 mlreactor. The temperature were set to 15° C. Then 2.40 g bromine (1:1Mmol bromine/mmol vinyl) was dissolved in 65 g sodium acetate buffer pH5 and added to the reactor. After 5 minutes the particles were washedwith 500 ml water.

Reaction with ammonia: 10 g particles were transferred to a 100 mlreactor with 54.4 g 25% ammonia solution. The temperature was increasedto 60° C. and the suspension was stirred mechanically at 300 rpm. After16 hours the mixture was washed with 700 ml of water an 300 ml of 20%ethanol. Amine: 0.30 mmol amine/g particles.

The amine content was found to be 0.30 mmol/gram particles by titrationof the hydrochloric acid amine salt by silver nitrate.

The aminated particles so obtained have been successfully used in solidphase oligonucleotide synthesis by conventional methods.

What is claimed is:
 1. A coated polymer article wherein the coating iscovalently bound to the polymer surface via double bonds of the polymer,comprising

a) P comprises a basic polymer and further groups—L—CH(—X(R₁)_(p))—CH₂(—Y(R₂)_(p)); b) L is a part of a pending group i)projecting from the basic polymer and ii) comprising a double bond thathas been utilized for introducing —X(R₁)_(p) and —Y(R₂)_(p); c) X and Yare independently selected from halogens, in particular Br, and N, S andO; d) R₁ is hydrogen, a straight, branched or cyclic alkyl or acylgroup, such as C₁-C₁₀ alkyl, C₁-C₁₈ acyl, or R₂ when X is N, O or S withparticular emphasis for X equals Y equals O; e) R₂ is hydrogen,straight, branched or cyclic alkyl such as C₁-C₁₈ alkyl, —R₃(—NH₂)_(q),alkylaryl or arylalkyl in which the alkyl part may contain 1-18 carbonatoms, —R₃(—NH—CR₄═O)_(q), —CR₄═O or poly lower alkyloxy groups that mayhave been terminally acylated or alkylated; f) p is an integer 0-3, withthe provision that when X is halogen p equals O, when X is O p equals 1,when X is S p equals 1 or 2, and when X is N p equals 1, 2 or 3, andwith the further provision that for p equals 2 or 3 the two or threegroups R₂ may be identical or different; g) R₃ is a straight, cyclic orbranched alkyl, —O-alkyl, hydroxyalkyl, phenylalkyl, with up to 18carbon atoms in the alkyl part, and R₄ is a straight, branched or cyclicalkyl group, such as C₁-C₁₈ alkyl; h) q is an integer >0 representingthat one or more hydrogens in R₃ may have been replaced with arespective primary amino group (—NH₂) or a respective —NH—CR₄═O group.2. A coated polymer article according to claim 1, wherein said basicpolymer of the article is cross-linked polystyrene-divinylbenzene and Lis a benzene ring.
 3. A coated polymer article according to claim 1,wherein said article is in the form of particles with a diameter of3-100 μm.
 4. Method of performing chromatographic separation, saidmethod comprising contacting the material to be separated with aseparation medium comprising said polymer article according to claim 1.5. Method of performing solid phase oligonucleotide synthesis, saidmethod comprising synthesizing oligonucleotides on a solid supportcomprising said polymer article according to claim 1, exhibiting primaryor secondary amino groups.
 6. Method of performing reverse phasechromatography said method comprising contacting the material to beseparated with a medium comprising said polymer article according toclaim 1.